Notch signaling is a highly conserved cell communication system that regulates a wide variety of cell types throughout development and into adulthood. Defects in Notch signal transduction have been implicated in a number of human disease states including cancer, stroke, and Alzheimer's disease, underscoring the importance of understanding the basic mechanisms of Notch signal transduction. Cell-surface Notch receptors, following engagement with cell-surface ligands, undergo a series of proteolytic cleavage events which release the intracellular domain of Notch. This proteolytic fragment translocates to the nucleus of the signal receiving cell where it directly regulates transcription of Notch target genes. Based on studies in Drosophila, a model for Notch signaling has been proposed in which the ligand-signaling cell plays an active role in Notch activation beyond ligand presentation. These models propose that during signal transduction the ligand cell actively removes the Notch extracellular domain through the poorly understood phenomenon of trans-endocytosis. This proposal aims to determine if trans-endocytosis is a characteristic of ligand- induced Notch signaling in mammals. To this end, a coculture system that can be examined by confocal microscopy has been developed. This coculture paradigm will allow characterization of the specifics of Notch transfer to Delta cells, providing insight into the molecular mechanism of trans-endocytosis and its relevance to Notch signaling. [unreadable] [unreadable] [unreadable]